Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Abstract

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.

Figure 1

Premature induction of autophagy induces midgut PCD. Morphology of midguts at 0 h RPF (left) showing (a) control (NP1-GAL4/+) compared with (b) Atg1 overexpression (NP1-GAL4/+ UAS-Atg1^6B/+) with premature contraction of gastric caeca (arrows). Histological analysis of paraffin sections at +12 h RPF (right) shows that at the later time point midgut contraction is similar in (a) control (NP1-GAL4/+) and (b) Atg1 overexpression (NP1-GAL4/+ UAS-Atg1^6B/+). Scale bars in (a) represent 200 μm. (c) Quantification of the length of the gastric caeca at 0 h RPF measured with Zeiss automeasure software from control (NP1-GAL4/+) and Atg1 overexpression (NP1-GAL4/+ UAS-Atg1^6B/+) (N=4 and 9, respectively). Quantification is average length (μm)±S.D. *P<0.001 (compared with the control). (d) Mosaic clones from feeding larvae expressing Atg1 marked by GFP (green) and DNA (blue) (hsFLP; mCherry-Atg8a/+ Act>CD2>GAL4, UAS-nlsGFP/UAS-Atg1^6B) have increased mCherry-Atg8a puncta (red) compared with the neighbouring cells and smaller cell size. Atg1 clone and neighbouring cell outlined. Scale bars in (d) represent 50 μm

Figure 2

Association of growth arrest with autophagy and midgut degradation. The localization of class I PI3K activity reporters, tGPH (a and b) and phospho-Akt (c), in midguts. (a) Cortical localization of tGPH (green) and DNA (blue) in midgut from feeding larvae at −8 h RPF (left, arrow) and following puparium formation +2 h RPF (middle) tGPH is no longer localized at the cortex. Inhibition of autophagy by (a) Atg1^IR knockdown (NP1-GAL4/+ UAS-Atg1^IR/+) and (b) in Atg2 mutant (Atg2/Df) display cortical localization of tGPH at +2 h RPF (arrow) compared with controls. Scale bar in (a) represents 25 μm in all panels in (a) and (b). (c) Cortical localization of phospho-Akt (green, arrow) adjacent to Fasciclin III (red), a cell surface marker, in control midgut from late larvae (−4 h RPF). Following puparium formation (+2 h RPF) phospho-Akt is redistributed from the cell cortex. Inhibition of autophagy by knockdown of Atg1^IR (NP1-GAL4/+ UAS-Atg1^IR/+) or Atg18^IR (NP1-GAL4/+ UAS-Atg18^IR/+) shows persistence of cortically localized phospho-Akt at +2 h RPF (arrow). Induction of autophagy by expression of Atg1 (NP1-GAL4/+ UAS-Atg1^6B/+) shows decreased cortically localized phospho-Akt (arrow). Scale bar is 30 μm in all panels in (c)

Figure 3

Maintenance of growth delays midgut removal. Mosaic clones of (a) Dp110 (hsFLP; mcherry-Atg8a/UAS-Dp110; Act>CD2>GAL4, UAS-nlsGFP) and (b) Ras^V12 (hsFLP; mcherry-Atg8a/+ Act>CD2>GAL4, UAS-nlsGFP/UAS-Ras^V12) from 0 h RPF gastric caeca marked by GFP (green) have decreased levels of mCherry-Atg8a puncta (red) compared with the neighbouring cells and larger cell size. A Dp110 or Ras^V12 clone and neighbouring cells are outlined. Scale bar in (a) represents 50 μm in all panels in (a) and (b). (c) Morphology of midguts at +4 h RPF showing control (left, NP1-GAL4/+) compared with Ras^V12 (right, NP1-GAL4/+ UAS-Ras^V12/+) showing persistent gastric caeca (arrow) compared with the absent gastric caeca in the control (arrow). (d) Histological analysis of paraffin sections at +12 h RPF of control (left, NP1-GAL4/+) and Ras^V12 (right, NP1-GAL4/+ UAS-Ras^V12/+) shows less condensed midgut compared with control. Scale bars in (c and d) represent 200 μm. (e) Quantification of gastric caeca size (average pixels±S.D.) at +4 h RPF from control (NP1-GAL4/+) and Ras^V12 overexpression (NP1-GAL4/+ UAS- Ras^V12/+ (N=8 and 7, respectively). *P<0.001 (compared with the control). (f) Caspase activity was measured from −4 h RPF larval lysates on DEVD-AMC, represented as pmol DEVD/min. The expression of p35 in the midgut inhibits the majority of caspase activity present in larvae. Data are mean from three independent experiments, with error bars representing S.E.M. *P<0.05 (compared with the control)

Figure 4

Autophagy inhibition delays midgut removal that is suppressed by inhibition of PI3K pathway. Morphology of midguts at 0 h RPF (left) and +4 h RPF (middle) showing (a) control (NP1-GAL4/+) compared with (b) PTEN expression (NP1-GAL4/+ UAS-PTEN/+) and (c) TSC1/2 expression (NP1-GAL4/+ UAS-TSC1, UAS-TSC2/+) showing decreased size with contracted gastric caeca at 0 h RPF and smaller midgut at +4 h RPF. Histological analysis of paraffin sections at +12 h RPF (right) shows significant midgut contraction in (b) PTEN expression and (c) TSC1/2 expression compared with control (a) (NP1-GAL4/+). (d) Inhibition of autophagy severely delays midgut degradation. Morphology of midguts at 0 h RPF (left) and +4 h RPF (middle) show a significant delay in midgut histolysis in (d) Atg1^IR (NP1-GAL4/+ UAS-Atg1^IR/+) compared with (a) control as seen by the presence of less-contracted gastric caeca at +4 h RPF (arrows). Histological analysis of paraffin sections at +12 h RPF (right) shows an enlarged midgut in (d) Atg1^IR compared with (a) control. The combined expression of (e) Atg1^IR PTEN and (f) Atg1^IR TSC1/2 suppressed the delay in midgut histolysis with greater contraction of gastric caeca at +4 h RPF (arrows) compared with (d) Atg1^IR alone. The delayed midgut histolysis in (d) Atg1^IR is reduced by co-expression of (e) PTEN or (f) TSC1/2 at +12 h RPF. Scale bars in (a) represent 200 μm in all panels. (g) Mosaic clones of PTEN (hsFLP; mCherry-Atg8a/+ Act>CD2>GAL4, UAS-nlsGFP/UAS-PTEN) marked by GFP (green) and DNA (blue) have smaller cell size with increased autophagy compared with the neighbouring cells. Scale bar in (g) is 50 μm. (h) Quantification of gastric caeca size (average pixels±S.D.) at 0 h RPF from control, PTEN, TSC1/2, Atg1^IR, Atg1^IR PTEN and Atg1^IR TSC1/2 (N=10, 12, 9, 8, 5 and 12, respectively). *P<0.001 (compared with the control). (i) Quantification of gastric caeca size (average pixels±S.D.) at +4 h RPF measured from control, PTEN, TSC1/2, Atg1^IR, Atg1^IR PTEN and Atg1^IR TSC1/2 (N=8, 8, 9, 9, 10 and 9, respectively). *P<0.001 (compared with the control). (j) Caspase activity was measured from −4 h RPF larval lysates on DEVD-AMC, represented as pmol DEVD/min. Data are mean from three independent experiments, with error bars representing S.E.M. *P<0.05 (compared with control)

Figure 5

Autophagy inhibition by Atg18 knockdown delays midgut removal that is suppressed by inhibition of PI3K pathway. Morphology of midguts at 0 h RPF (left) and +4 h RPF (middle) show a significant delay in midgut histolysis in (a) Atg18^IR (NP1-GAL4/+ UAS-Atg18^IR/+) as seen by the presence of less-contracted gastric caeca at +4 h RPF (arrows). Histological analysis of paraffin sections at +12 h RPF (right) shows an enlarged midgut in (a) Atg18^IR. The combined expression of (b) Atg18^IR PTEN and (c) Atg18^IR TSC1/2 suppressed the delay in midgut histolysis with greater contraction of gastric caeca at +4 h RPF (arrows) compared with (a) Atg18^IR alone. The delayed midgut histolysis in (a) Atg18^IR is reduced by co-expression of (b) PTEN or (c) TSC1/2 at +12 h RPF. Scale bars represent 200 μm. (d) Quantification of gastric caeca size (average pixels±S.D.) at 0 h RPF from control, PTEN, TSC1/2, Atg18^IR, Atg18^IR PTEN and Atg18^IR TSC1/2 (N=10, 12, 9, 7, 6 and 10, respectively). *P<0.001 (compared with the control). (e) Quantification of gastric caeca size (average pixels±S.D.) at +4 h RPF from control, PTEN, TSC1/2, Atg18^IR, Atg18^IR PTEN and Atg18^IR TSC1/2 (N=8, 8, 9, 9, 5 and 6, respectively). *P<0.001 (compared with the control)